Many experimental and associative studies indicate decreased insulin activity In specific regions of brain as a potential contributor to Alzheimer's disease (AD) and perhaps related neurodegenerative diseases. Importantly, recent pilot clinical trials have concluded that augmenting central nervous system (CNS) insulin activity improves or protects cognitive function in patients with amnestic Mild Cognitive Impairment (aMCl) or mild AD, perhaps especially patients without an e4 allele of the apolipoprotein E gene {APOE). We hypothesize that specific insulin-induced changes in the human cerebrospinal fluid (CSF) proteome will illuminate a set of biomarkers responsive to alterations In CNS Insulin activity. We will this hypothesis by achieving the following specific aims: (i) discover, confirm, and validate a novel CNS Insulin-Responsive Multi-Analyte Profile (MAP) of specific changes In the CSF proteome with Increased CNS insulin activity using samples from two placebo-controlled randomized trials of intranasal insulin in patients with aMCI/mild AD, including Project 2, (ii) determine the significance of decreased CNS insulin activity In age-related cognitive decline, pre-clinical AD, and related diseases using our CNS Insulin-Responsive MAP, CSF samples and cognitive testing data from our Clinical Core and collaborative UWADRC CSF Bank, and established AD CSF biomarkers from our Neuropathology and Targeted Molecular Testing (NP&TMT) Core, and (iii) quantify brain regional concentration and cellular distribution of selected proteins from our CNS Insulin-Responsive MAP in a large series of autopsies from Controls and patients with AD who did or did not have diabetes mellitus (DM) whose tissue already is in the NP&TMT Core. When successfully completed, our proposed studies will have generated new knowledge about the mechanisms of action of increased CNS insulin activity in patients with aMCI/mild AD receiving insulin therapy, inversely determined the functional significance and specificity of decreased CNS insulin activity In age-related cognitive decline, preclinical AD, and related neurodegenerative diseases, and explored the regional and cellular distribution of selected key protein candidates in a well-characterized autopsy series of AD patients and controls with and without DM.